Packing material for high-performance liquid chromatography

ABSTRACT

There is provided a packing material having a carrier coated with a substance having a separating capacity for high-performance liquid chromatography, wherein the performance of the packing material can be sufficiently exhibited with little dispersion of separating capacity thereof. The packing material contains part of a coating solvent remaining therein, whereby the excellent separating capacity of the packing material can be exhibited.

TECHNICAL FIELD TO WHICH THE INVENTION PERTAINS

The present invention relates to a packing material for high-performanceliquid chromatography, and provides a packing material having anexcellent separating capacity with little dispersion particularly in thefield of separation of optical isomers.

PRIOR ART

There are a variety of packing materials for high-performance liquidchromatography, examples of which include one comprising a granulated(crushed or bead-like) substance having a separating capacity in itself,one having such a substance chemically bonded to a carrier, and onehaving a carrier coated with such a substance. Particularly in the fieldof separation or optical isomers, a packing material having a carriercoated with an optically active substance having the capacity ofseparation of optical isomers is actually used in many cases. Knownexamples of such a packing material include one having a carrier coatedwith an optically active crown ether compound as a low-molecularcompound (JP-A 62-210053), one having a carrier coated with an opticallyactive triphenylmethyl methacrylate polymer as a synthetic polymer (JP-A57-150432), one having a carrier coated with cellulose triacetate as aderivative of a polysaccharide (JP-A 60-82858), one having a carriercoated with cellulose benzoate (JP-A 60-40952), and one having a carriercoated with cellulose phenylcarbamate (JP-A 60-108751), which arecommercialized and widely used by virtue of their high optical resolvingpowers.

However, columns packed with such a packing material, even if packedwith the same packing material, sometimes cause a large difference inseparating performance, sometimes a good separation but sometimes a poorseparation depending on different production lots of the packingmaterial, when some racemic compounds as an object of separation areused. Particularly in the case of preparation (or aliquot), whenseparation is poor, it is unavoidable to resort to a low-efficiencyseparating operation by, for example, decreasing the flow rate of amobile phase into the column or decreasing the feed rate of a sample.

With a view to coping with the foregoing problems, an object to besolved by the present invention is to provide a packing material havinga carrier coated with a substance having a separating capacity forhigh-performance chromatography, wherein the capacity of the packingmaterial can be sufficiently exhibited with little dispersion ofseparating capacity thereof.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 illustrates the results of the optical resolution tests carriedout in Examples 1 and 2 and Comparative Example 1.

DISCLOSURE OF THE INVENTION

As a result of intensive investigations, the inventors of the presentinvention have found out that the separating capacity of a packingmaterial having a carrier coated with a substance having a separatingcapacity for high-performance liquid chromatography is affected by theamount of a coating solvent remaining in the packing material. Thepresent invention has been completed based on this finding.

The present invention provides a packing material for high-performanceliquid chromatography which has a carrier coated with a substance havinga separating capacity and wherein part of the coating solvent remains.

It is preferred that the substance having a separating capacity be anoptically active triphenylmethyl methacrylate polymer, an opticallyactive (meth)acrylic amide polymer or a derivative of a polysaccharide,that the substance having a separating capacity be a derivative of apolysaccharide, that the substance having a separating capacity be anester derivative, carbamate derivative or ether derivative of apolysaccharide, or that the substance having a separating capacity be anaromatic ester derivative of a polysaccharide.

It is further preferred that the substance having a separating capacitybe an aromatic ester derivative of a polysaccharide, wherein 30 to 100%on the average of the hydroxyl groups of the polysaccharide aresubstituted by a group represented by the following formula (I):##STR1## wherein R¹ to R⁵ are each hydrogen atom, a C₁ to C₈ alkylgroup, a C₁ to C₈ alkoxyl group, a C₆ to C₁₄ aromatic group, or ahalogen atom.

Among the substance having a separating capacity, cellulosetris(4-methylbenzoate) is preferred.

It is preferred that the coating solvent be a mixture of a low-boilingsolvent with a high-boiling solvent, and that the amount of thelow-boiling solvent remaining in the packing material be at most 1.5 %by weight, while the amount of the high-boiling solvent remaining in thepacking material be 15 to 19% by weight.

It is also preferred that the substance having a separating capacity bea carbamate derivative of a polysaccharide, or that the substance havinga separating capacity be an aromatic carbamate derivative of apolysaccharide.

It is further preferred that the substance having a separating capacitybe an aromatic carbamate derivative of a polysaccharide, wherein 30 to100% on the average of the hydroxyl groups of the polysaccharide aresubstituted by a group represented by the following formula (II):##STR2## wherein R¹ to R⁵ are each hydrogen atom, a C₁ to C₈ alkylgroup, a C₁ to C₈ alkoxyl group, a C₆ to C₁₄ aromatic group, or ahalogen atom.

Among the substance having a separating capacity, cellulosetris(4-chlorophenylcarbamate) is preferred.

It is preferred that the amount of the remaining coating solvent be 4 to22% by weight, and that the coating solvent be acetone, methylenechloride, dichloromethane, tetrahydrofuran, or dioxane.

It is desired that the low-boiling solvent and the high-boiling solventbe methylene chloride and methyl benzoate, respectively, and that theamount of the remaining coating solvent be 10 to 25% by weight.

The present invention further provides a method of separating an opticalisomer by high-performance liquid chromatography, wherein use is made ofa packing material which has a carrier coated with a substance having aseparating capacity and in which part of a coating solvent remains; anda high-performance liquid chromatography column packed with a packingmaterial which has a carrier coated with a substance having a separatingcapacity and in which part of a coating solvent remains.

In the present invention, any substance may be used as the substancehaving a separating capacity in so far as it has a capacity ofseparation of liquid mixture. Examples of that substance include anoptically active triphenylmethyl methacrylate polymer, an opticallyactive (meth)acrylic amide polymer, and derivatives of polysaccharides,among which derivatives of polysaccharides are preferred.

Any polysaccharide may be used as the starting material of a derivativeof the polysaccharide irrespective of whether it is a syntheticpolysaccharide, a natural polysaccharide or a modified naturalpolysaccharide in so far as it has optical activity, thoughpolysaccharides having a high regularity of bonding mode are preferred.Examples of the polysaccharide include β-1,4-glucan (cellulose),α-1,4-glucan (amylose, amylopectin), α-1,6-glucan, β-1,4-galactan,β-1,6-glucan (pustulan), β-1,3-glucan (e.g., curdlan, schizophyllan),α-1,3-glucan, β-1,4-mannan, α-1,6-mannan, β-1,2-fructan (inulin),β-2,6-fructan (levan), β-1,4-xylan, β-1,4-chitosan,β-1,4-N-acetylchitosan (chitin), pullulan, agarose, and alginic acid.Other examples include starch containing amylose and cyclodextrins ascyclic polysaccharides. Especially preferred are β-1,4-glucan(cellulose), α-1,4-glucan (amylose, amylopectin), β-1,4-chitosan,β-1,4-N-acetylchitosan (chitin), β-1,4-mannan, β-1,4-xylan, inulin,curdlan, and cyclodextrins, since they can each be easily obtained inthe form of a high-purity polysaccharide. The number-average degree ofpolymerization (the average number of pyranose rings or furanose ringscontained in one molecule) of the polysaccharide is at least 2,preferably at least 5, with no particular upper limit, though it ispreferably at most 500 because of easy handleability.

Kinds of derivatives of polysaccharide usable in the present inventioninclude ester derivatives, carbamate derivatives, and ether derivatives,among which ester derivatives and carbamate derivatives are preferred.Further preferred are aromatic ester derivatives and aromatic carbamatederivatives.

An ordinary reaction of an alcohol with an acid chloride or an acidbromide for ester formation can be applied as such to the synthesis ofan aromatic ester derivative of a polysaccharide, which can be obtained,for example, by reacting the polysaccharide with the corresponding acidchloride or acid bromide in a suitable solvent in the presence of aLewis base such as a tertiary amine.

An ordinary reaction of an alcohol with an isocyanate for urethaneformation can be applied as such to the synthesis of an aromaticcarbamate derivative of a polysaccharide, which can be obtained, forexample, by reacting the polysaccharide with an isocyanate having thecorresponding aromatic ring in a suitable solvent in the presence of aLewis base such as a tertiary amine or a Lewis acid such as a tincompound as the catalyst. Meanwhile, the isocyanate can be easilysynthesized, for example, by reacting the amino group of thecorresponding aniline derivative with phosgene.

The carrier to be used in the present invention may be any of organicand inorganic carriers, preferably an inorganic carrier. Suitableexamples of the inorganic carrier include silica gel, alumina, magnesia,titanium oxide, glass, silicates, and kaolin, among which silica gel isespecially preferred. The particle size of the carrier, though variesdepending on the size of a column to be used, is generally 1 μm to 10mm, preferably 1 μm to 300 μm. The carrier is preferably porous in anaspect of properties. In this case, the average pore size of the carrieris 10 Å to 100 μm, preferably 50 Å to 50,000 Å. The amount of thederivative of the polysaccharide that may be supported on the carrier is1 to 100% by weight, preferably 5 to 50% by weight, based on thecarrier.

According to a method of supporting the aromatic ester derivative orcarbamate derivative of the polysaccharide on the carrier, the aromaticester derivative or carbamate derivative of the polysaccharide isdissolved in solvent to prepare a dope, which is then dropped little bylittle into the carrier under stirring to uniformly coat the carriertherewith, and the coated carrier, after the completion of coating, isheated and dried under reduced pressure or in an air stream to removethe solvent.

When the solvent to be used for supporting the aromatic ester derivativeof the polysaccharide on the carrier, i.e., the coating solvent, is asolvent mixture of a low-boiling solvent capable of well dissolving thearomatic ester derivative of the polysaccharide with a high-boilingsolvent capable of swelling the aromatic ester derivative of thepolysaccharide, it is possible to secure a good separating capacity.Usable low-boiling solvents include methylene chloride (boiling point:40° C.) and the like, while usable high-boiling solvents include methylbenzoate (boiling point: 200° C.) and the like.

Examples of the solvent to be used in supporting the carbamatederivative of the polysaccharide on the carrier include acetone,methylene chloride, dichloromethane, tetrahydrofuran and dioxane.

In the present invention, it is a matter of importance that part of thecoating solvent be let to remain in the packing material in the step ofremoving the coating solvent by drying.

In the case of the aromatic ester derivative of the polysaccharide, itis preferred that the amount of the solvent remaining in the packingmaterial be 10 to 25% by weight as a whole. When it exceeds 25% byweight, the mutual agglomeration of particles occurs to make the packingmaterial unusable. In a packing material having the carrier coated withthe aromatic ester derivative of the polysaccharide, it is preferredthat the amount of the low-boiling solvent remaining in the packingmaterial be at most 1.5% by weight, and that the amount of thehigh-boiling solvent remaining in the packing material be 15 to 19% byweight. When the amounts of the solvents remaining in the packingmaterial are within these ranges, the packing material exhibits a goodseparating capacity. In order that the amounts of the solvents remainingin the packing material fall within these ranges, drying conditionsafter coating, i.e., temperature, pressure, time, etc., are determinedexperimentally, and the packing material is produced under thedetermined drying conditions.

In the case of an aromatic carbamate derivative of a polysaccharide, itis preferred that the amount of the remaining solvent be 4 to 22% byweight.

The customary slurry packing method is used as the method of packing theresulting packing material with a column. In this method, n-hexane,2-propanol, methanol or the like is used as the solvent.

The packing material of the present invention for high-performanceliquid chromatography, wherein part of a coating solvent remains, cansufficiently exhibit its capacity with little dispersion of separatingcapacity. Particularly in the case of preparation (or aliquot), theproblem of a low-efficiency separating operation caused by a decrease inthe flow rate of a mobile phase into a column and a decrease in the feedrate of a sample, which has hitherto been unavoidable in the case ofpoor separation, can be solved by the present invention.

EXAMPLES

The following Examples will specifically illustrate the presentinvention, though it should not be construed as limiting the scope ofthe present invention.

Example 1

10 g of cellulose tris(4-methylbenzoate) was dissolved in a solventmixture of 40 ml of methylene chloride and 10 ml of methyl benzoate toprepare a viscous dope, which was then dropped little by little into 40g of silica gel treated with aminopropylsilane carbamoylated with3,5-dimethylphenyl isocyanate (manufactured by Daiso Co., Ltd., averageparticle size: 20 μm, average micropore size: 1300 Å) under stirring at25° C. After the completion of dropping, methylene chloride was mainlydistilled away under further stirring first under 500 Torr. at 40° C.for 45 minutes and then under 100 Torr. at 40° C. for 1 hour.Thereafter, the packing material was dispersed in 200 ml of methanol togive a slurry, which was then filtered to recover a solid material. Therecovered solid material was then dried under 2 to 5 Torr. at 60° C. for3 hours to give a packing material. The results of measurement of theamounts of the solvents remaining in this packing material are shown inTable 1.

Example 2

Substantially the same procedure as in Example 1 except that the finaldrying time in Example 1 was changed to 2.5 hours was repeated toprepare a packing material. The results of measurement of the amounts ofthe solvents remaining in this packing material are shown in Table 1.

Comparative Example 1

Substantially the same procedure as in Example 1 except that the finaldrying time in Example 1 was changed to 2 hours was repeated to preparea packing material. The results of measurement of the amounts of thesolvents remaining in this packing material are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Amounts of Solvents Remaining in Packing Material                             (wt. %)                                                                                  Packing material                                                   Solvent      Ex. 1      Ex. 2   Comp. Ex. 1                                   ______________________________________                                        methylene chloride                                                                         0.22       1.07    1.72                                          methyl benzoate                                                                            17.9       18.0    18.0                                          ______________________________________                                    

Application Example A

Each of the packing material prepared in Example 1, Example 2 andComparative Example 1 was fed into a stainless steel column (innerdiameter: 1.0 cm, length: 25 cm) by the slurry method wherein ann-hexane/2-propanol (volume ratio 7/3) was used as a solvent mixture.With this column, the optical resolution experiments of racemicmodification of cisapride was carried out under the followingconditions. The experimental results (separation factor andchromatogram) are shown in Table 2.

mobile phase: n-hexane/2-propanol (volume ratio 7/3) containing 0.1% ofdiethylamine

flow rate: 4.7 ml/min

detection: ultraviolet detector, 254 nm

(sensitivity: 0.16 AFUS)

temperature: 25° C.

amount of feed: 50 μg

Application Example B

Each of the packing material prepared in Example 1, Example 2 andComparative Example 1 was fed into a stainless steel column (innerdiameter: 1.0 cm, length: 25 cm) by the slurry method wherein ann-hexane/2-propanol (volume ratio 7/3) was used as a solvent mixture.With this column, the optical resolution experiments of racemicmodification of indapamide was carried out under the followingconditions. The experimental results (separation factor andchromatogram) are shown in Table 2. Table 2 has been presented asFIG. 1. A reference to Table 2 is the same as a reference to FIG. 1.

mobile phase: n-hexane/2-propanol (volume ratio 7/3) containing 0.1% ofdiethylamine

flow rate: 4.7 ml/min

detection: ultraviolet detector, 254 nm

(sensitivity: 0.16 AFUS)

temperature: 25° C.

amount of feed: 50 μg

Example 3

10 g of cellulose tris(4-chlorophenylcarbamate) was dissolved in 65 mlof acetone to prepa re a viscous dope. This dope was then dropped littleby little into 40 g of silica gel treated with aminopropylsilane(manufactured by Daiso Co., Ltd., average particle size: 20 μm, averagemicropore size: 1300 Å) under stirring at 25° C. The system was adjustedto a reduced pressure (350 Torr.) under further stirring, followed bydistilling acetone away at a temperature of 45° C for 1 hour.Thereafter, the packing material was dispersed in 250 ml of 2-propanolto give a slurry, which was then filtered to recover a solid material.The solid material thus obtained was dried under 2 to 5 Torr. at 60° C.for 3 hours to give a packing material. The result of measurement of theamount of the solvent remaining in this packing material is shown inTable 3.

                  TABLE 3                                                         ______________________________________                                                 Amt. of                                                                              Results of expt. on opt.                                               remaining                                                                            resoln.                                                                coating                                                                              retentn.  separatn.                                                    solvent                                                                              vol.      factor   resoln.                                             (wt. %)                                                                              (k'.sub.1)                                                                              (α)                                                                              (Rs)                                       ______________________________________                                        Ex. 3      20.1     1.89      3.10   3.17                                     Ex. 4      10.8     2.18      3.17   3.22                                     Ex. 5       4.7     1.96      3.22   3.34                                     Comp. Ex. 2                                                                              27.6     2.02      3.09   2.91                                     Comp. Ex. 3                                                                               3.3     2.14      3.34   2.70                                     ______________________________________                                    

Examples 4 and 5 and Comparative Examples 2 and 3

Substantially the same procedure as in Example 3 except that the finaldrying time in Example 3 was varied was repeated to prepare a variety ofpacking material as shown in Table 3.

Application Example C

Each of the packing material prepared in Examples 3, 4 and 5 andComparative Examples 2 and 3 was fed into a stainless steel column(inner diameter: 1.0 cm, length: 25 cm) by the slurry method wherein an2-propanol was used. With this column, the optical resolutionexperiments of racemic modification of disopyramide was carried outunder the following conditions. The experimental results are shown inTable 3.

mobile phase: n-hexane/2-propanol (volume ratio 1/1) containing 0.1% ofdiethylamine

flow rate: 4.7 ml/min

detection: ultraviolet detector, 254 nm

(sensitivity: 0.16 AFUS)

temperature: 25° C.

amount of feed: 50 μl (1,000 ppm)

As can be seen in Table 3, the resolution was low and so unsuitable forpreparation of disopyramide in Comparative Examples 2 and 3 wherein theamount of the remaining acetone as the coating solvent departed from therange of 4 to 22% by weight.

Example 6

10 g of cellulose tris(4-chlorophenylcarbamate) was dissolved in 65 mlof acetone to prepare a viscous dope. This dope was then dropped littleby little into 40 g of silica gel treated with aminopropylsilane(manufactured by Daiso Co., Ltd., average particle size: 50 μm, averagemicropore size: 1000 Å) under stirring at 25° C. The system was adjustedto a reduced pressure (350 Torr.) under further stirring, followed bydistilling acetone away at a temperature of 45° C. for 1 hour.Thereafter, the resulting packing material was dispersed in 250 ml of2-propanol to give a slurry, which was then filtered to recover a solidmaterial. The solid material thus obtained was dried under 2 to 5 Torr.at 60° C. for 3 hours to give a packing material. The result ofmeasurement of the amount of the solvent remaining in this packingmaterial is shown in Table 4.

                  TABLE 4                                                         ______________________________________                                                 Amt. of                                                                              Results of expt. on opt.                                               remaining                                                                            resoln.                                                                coating                                                                              retentn.  separatn.                                                    solvent                                                                              vol.      factor   resoln.                                             (wt. %)                                                                              (k'.sub.1)                                                                              (α)                                                                              (Rs)                                       ______________________________________                                        Ex. 6      18.2     3.81      1.31   0.71                                     Ex. 7       4.4     4.30      1.29   0.61                                     Comp. Ex. 4                                                                              23.1     3.54      1.22   0.31                                     Comp. Ex. 5                                                                               1.5     3.96      1.17   0.21                                     ______________________________________                                    

Example 7 and Comparative Examples 4 and 5

Substantially the same procedure as in Example 6 except that the finaldrying time in Example 6 was varied was repeated to prepare a variety ofpacking material as shown in Table 4.

Application Example D

Each of the packing material prepared in Examples 6 and 7 andComparative Examples 4 and 5 was fed into a stainless steel column(inner diameter: 1.0 cm, length: 25 cm) by the slurry method wherein2-propanol was used. With this column, the optical resolutionexperiments of racemic modification of perisoxal was carried out underthe following conditions. The experimental results are shown in Table 4.

mobile phase: n-hexane/2-propanol (volume ratio 9/1) containing 0.1% ofdiethylamine

flow rate: 4.7 ml/min

detection: ultraviolet detector, 254 nm

(sensitivity: 0.16 AFUS)

temperature: 25° C.

amount of feed: 50 μl (1,000 ppm)

As can be seen in Table 4, the resolution was low and so unsuitable forpreparative chromatography of perisoxal in Comparative Examples 4 and 5wherein the amount of the remaining acetone as the coating solventdeparted from the range of 4 to 22% by weight.

We claim:
 1. A packing material for high-performance liquidchromatography which has a carrier coated with a substance having aseparating capacity and wherein part of a coating solvent remains and isa mixture of a low-boiling solvent and a high-boiling solvent, and theamount of the low-boiling solvent remaining in the packing is at most1.5% by weight, while the amount of the high-boiling solvent remainingin the packing material is 15 to 19% by weight.
 2. The packing materialas claimed in claim 1, wherein the substance having a separatingcapacity is an optically active triphenylmethyl methacrylate polymer, anoptically active (meth)acrylic amide polymer, or a derivative of apolysaccharide.
 3. The packing material as claimed in claim 1, whereinthe substance having a separating capacity is a derivative of apolysaccharide.
 4. The packing material as claimed in claim 1, whereinthe substance having a separating capacity is an ester derivative,carbamate derivative or ether derivative of a polysaccharide.
 5. Thepacking material as claimed in claim 1, wherein the substance having aseparating capacity is an aromatic ester derivative of a polysaccharide.6. The packing material as claimed in claim 1, wherein the substancehaving a separating capacity is an aromatic ester derivative of apolysaccharide, wherein 30 to 100% on the average of the hydroxyl groupsof the polysaccharide are substituted by a group represented by thefollowing formula (I): ##STR3## wherein R¹ to R⁵ are each hydrogen atom,a C₁ to C₈ alkyl group, a C₁ to C₈ alkoxyl group, a C₆ to C₁₄ aromaticgroup, or a halogen atom.
 7. The packing material as claimed in claim 1,wherein the substance having a separating capacity is cellulosetris(4-methylbenzoate).
 8. The packing material as claimed in claim 1,wherein the substance having a separating capacity is a carbamatederivative of a polysaccharide.
 9. The packing material as claimed inclaim 1, wherein the substance having a separating capacity is anaromatic carbamate derivative of a polysaccharide.
 10. The packingmaterial as claimed in claim 1, wherein the substance having aseparating capacity is an aromatic carbamate derivative of apolysaccharide, wherein 30 to 100% on the average of the hydroxyl groupsof the polysaccharide are substituted by a group represented by thefollowing formula (II): ##STR4## wherein R¹ to R⁵ are each hydrogenatom, a C₁ to C₈ alkyl group, a C₁, to C₈ alkoxyl group, a C₆ to C₁₄aromatic group, or a halogen atom.
 11. The packing material as claimedin claim 1, wherein the substance having a separating capacity iscellulose tris(4-chlorophenylcarbamate).
 12. The packing material asclaimed in claim 1, wherein the low-boiling solvent is methylenechloride, while the high-boiling solvent is methyl benzoate.
 13. Amethod of separating an optical isomer by high-performance liquidchromatography, wherein use is made of the packing material of claim 1.14. A high-performance liquid chromatography column, packed with thepacking material of claim 1.